PROJECT SUMMARY Emerging evidence links neurological diseases, including retinal neuropathies, to defects in RNA biogenesis and processing. Cells in the retina may be particularly vulnerable to defects in RNA processing because of the high levels of transcription and splicing that are required for healthy visual function. Surprisingly, the very act of transcription itself can generate harmful structures termed R-loops that form when the nascent RNA transcript hybridizes with the double-stranded DNA. Persistent R-loops block transcription elongation and also expose the unprotected DNA to damage. Although once thought to be rare, R-loops can form quite abundantly, and cells possess mechanisms both to prevent R-loop formation, and to resolve persistent R-loops. Importantly, R-loops have recently been detected in human and mouse photoreceptors, leading to the critical question of how R-loop formation affects photoreceptor health. Mutations that increase R-loop formation have been associated with several neurodegenerative diseases, including glaucoma. Here, we hypothesize that accumulation of harmful R- loops in old photoreceptors results in DNA damage and inhibition of transcription, leading to cell death. To characterize how R-loops impact photoreceptor heath and function, we will modulate R-loop levels in the genetically tractable model system Drosophila. Increasing levels of RNAseH1 in old flies should resolve R-loops, reducing their accumulation and suppressing R-loop-induced DNA damage and gene expression defects. In contrast, reducing expression of the THO/TREX complex that prevents R-loop formation should increase R-loop levels in young flies, resulting in premature aging and retinal degeneration. Together these studies will address one of the key unresolved questions in eye biology: how does aging increase the susceptibility to ocular disease? This R21 application will explore the idea that the accumulation of persistent R-loops in aging neurons provides an intrinsic stress that contributes to the increased risk of ocular disease with age. These studies will pave the way for potential neuronal therapies targeted to reduce R-loop levels in aging photoreceptors, resetting their epigenome and thereby reducing their risk of retinal degeneration.